Methods of screening for compounds that decrease phosphorylation of merlin and may be useful in cancer treatment

ABSTRACT

The present inventions features methods for treating or preventing cancer (e.g., cancer of the central nervous system) by administering a compound that inhibits PAK kinase activity and/or merlin phosphorylation to a mammal (e.g., a human). The invention also provides screening methods for identifying additional inhibitors of PAK kinase activity and/or merlin phosphorylation.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the National Stage of International Application No.PCT/USO2/25568, filed August 13, 2002, which was published in Englishunder PCT Article 2 1(2), which claims benefit of U.S. ProvisionalApplication No. 60/311,873, filed Aug. 13, 2001, each of which is herebyincorporated by reference.

BACKGROUND OF THE INVENTION

Neurofibromatosis type 2 is an inherited disorder characterized by thedevelopment of Schwann cell tumors of the eighth cranial (auditory)nerve. Mutations and loss of heterozygosity of the NF2 locus have beendetected in various familial and sporadic tumors of the nervous system,including schwannomas, meningiomas, and ependymomas. These mutationshave been detected both in the germ-line of Nf2 patients andsporadically occurring tumors, indicative of a classical tumorsuppressor gene pattern. Together, these tumors account forapproximately 30% of central nervous system neoplasms in adults. Infurther support of a role for NF2 in tumor suppression, miceheterozygous for a Nf2 mutation are predisposed to a wide variety oftumors with high metastatic potential. In a separate model in which Nf2was inactivated specifically in Schwann cells, mice developedschwannomas and Schwann cell hyperplasia.

The longest and predominant splice form of the Nf2 gene codes for a595-amino acid protein called merlin that is highly similar to the band4.1 family of proteins. It is most closely related to the ERMproteins—ezrin, radixin, and moesin. The ERM proteins are thought tofunction as cell membrane-cytoskeleton linkers and are localized tocortical actin structures near the plasma membrane such as microvilli,membrane ruffles, and lamellipodia. Likewise, merlin is localized tocortical actin structures in patterns that partially overlap with theERMs. It has been proposed that intramolecular binding of the N-terminaland C-terminal domains conformationally regulates the ERM proteins bymasking binding sites for interacting proteins. The ERMs can also formhomo-dimers and hetero-dimers among themselves and with merlin, addingan additional level of complexity to the regulation of these proteins.The recently solved crystal structure of moesin N/C-terminal complexstrengthens this model of conformational regulation.

Unfortunately, many of the current treatments that destroy cancerouscells also affect normal cells, resulting in a variety of possibleside-effects, such as nausea, vomiting, low blood cell counts, increasedrisk of infection, hair loss, and ulcers in mucous membranes. Thus,improved methods are needed for the treatment and prevention of cancers,such as cancers of the nervous system.

SUMMARY OF THE INVENTION

In general, the invention provides novel methods for the treatment orprevention of cancer (e.g., cancer of the central nervous system) byadministering one or more compounds that inhibit PAK kinase activityand/or phosphorylation of merlin. Exemplary diseases that can be treatedor prevented using these methods include neurofibromatosis type 2 or anyother disease that involves aberrations in the function of the Nf2 geneor in the function of merlin.

In one aspect, the invention provides a method of treating, stabilizing,or preventing cancer in a mammal (e.g., a human) that involves reducingPAK kinase activity in the mammal. In desirable embodiments, a compoundthat reduces PAK kinase activity (e.g., PAK1, PAK2, PAK3, PAK4, PAK5,and/or PAK6 kinase activity) is administered to the mammal in an amountsufficient to treat, stabilize, or prevent cancer in the mammal.Desirably, an activity of a PAK kinase is reduced by at least 5, 10, 20,30, 40, 50, 60, or 80, 90, 95, or 100%. In various embodiments, thecompound is a purified or unpurified synthetic organic molecule,naturally occurring organic molecule, nucleic acid molecule, PAK kinaseantisense nucleic acid or double stranded RNA molecule, biosyntheticprotein or peptide, naturally occurring peptide or protein, PAK kinaseantibody, or dominant negative PAK kinase protein (e.g., a mutant orfragment of a PAK kinase). In other embodiments, the compound is anautoinhibitory region of a PAK kinase, such as a protein that includesor consists of at least 25, 50, 75, 100, 125, or 150 contiguous aminoacids of residues 82-146 of PAK2 or the corresponding region in anotherPAK kinase (e.g., residues 83-149 of PAK1). In certain embodiments, theprotein includes at least 25, 50, 75, 100, 125, 150, 200, or 300 of theN-terminal amino acids of a PAK kinase. In some embodiments, thecompound is staurosporine or an ATP analog.

In another aspect, the invention provides a method of treating,stabilizing, or preventing cancer in a mammal (e.g., a human) thatinvolves reducing the amount of merlin that is phosphorylated (e.g.,phosphorylation on serine 518) in the mammal. In desirable embodiments,a compound that reduces the phosphorylation level of merlin (e.g.,phosphorylation at serine 518) is administered to the mammal in anamount sufficient to treat, stabilize, or prevent cancer in the mammal.In other desirable embodiments, the amount of merlin that isphosphorylated at serine 518 is reduced by at least 5, 10, 20, 30, 40,50, 60, or 80, 90, 95, or 100%. In various embodiments, at least 20, 40,50, 60, 80, 90, or 95% of merlin is located in microvilli. Exemplarymerlin proteins have an amino acid sequence that is at least 40, 50, 60,70, 80, 90, 95, or 100% identical to the sequence of a region of humanmerlin or the sequence of full-length human merlin (accession numberP35240). In various embodiments, the compound is a purified orunpurified synthetic organic molecule, naturally occurring organicmolecule, nucleic acid molecule, PAK kinase antisense nucleic acid ordouble stranded RNA molecule, biosynthetic protein or peptide, naturallyoccurring peptide or protein, PAK kinase antibody, or dominant negativePAK kinase protein (e.g., a mutant or fragment of a PAK kinase). Inother embodiments, the compound is an autoinhibitory region of a PAKkinase, such as a protein that includes or consists of at least 25, 50,75, 100, 125, or 150 contiguous amino acids of residues 82-146 of PAK2or the corresponding region in another PAK kinase (e.g., residues 83-149of PAK1). In certain embodiments, the protein includes at least 25, 50,75, 100, 125, 150, 200, or 300 of the N-terminal amino acids of a PAKkinase. In some embodiments, the compound is staurosporine or an ATPanalog. In other embodiments, the compound is an anti-merlin antibody.Desirably, the amount of phosphorylated merlin that is bound by theantibody is at least 2, 5, 10, or 15-fold greater that the amount ofunphosphorylated merlin that is bound.

The invention also features methods for identifying or selectingcompounds that decrease PAK kinase activity or decrease the level ofmerlin phosphorylation and thus are useful for treating or preventingcancer in a mammal (e.g., a human).

Accordingly, in one aspect, the invention features a screening methodfor determining whether a compound is useful for treating, stabilizing,or preventing cancer in a mammal. This method involves measuring PAKkinase activity in a cell, tissue, or mammal in the presence and absenceof the compound. The compound is determined to treat, stabilize, orprevent cancer if the compound decreases PAK kinase activity. In someembodiments, the method also includes administering the compound to amammal in need of the treatment (e.g., a mammal with cancer or anincreased risk for cancer). In certain embodiments, the compound is amember of a library of at least 5, 10, 15, 20, 30, 50, or morecompounds, all of which are simultaneously administered to the cell,tissue, or mammal. In various embodiments, the compound is a purified orunpurified synthetic organic molecule, naturally occurring organicmolecule, nucleic acid molecule, PAK kinase antisense nucleic acid ordouble stranded RNA molecule, biosynthetic protein or peptide, naturallyoccurring peptide or protein, PAK kinase antibody, or dominant negativePAK kinase protein (e.g., a mutant or fragment of a PAK kinase). Inother embodiments, the compound is an autoinhibitory region of a PAKkinase, such as a protein that includes or consists of at least 25, 50,75, 100, 125, or 150 contiguous amino acids of residues 82-146 of PAK2or the corresponding region in another PAK kinase (e.g., residues 83-149of PAK1). In certain embodiments, the protein includes at least 25, 50,75, 100, 125, 150, 200, or 300 of the N-terminal amino acids of a PAKkinase. In some embodiments, the compound is staurosporine or an ATPanalog. In desirable embodiments, the compound decreases an activity ofa PAK kinase (e.g., the phosphorylation of merlin), the level of a PAKkinase mRNA or protein, the half-life of a PAK kinase mRNA or protein,the binding of a PAK kinase to a substrate or to another molecule, orthe level or activity of a protein that phosphorylates a PAK kinase.Desirably, the level of a PAK kinase mRNA or protein, an activity of aPAK kinase, the half-life of a PAK kinase mRNA or protein, the bindingof a PAK kinase to another molecule, or the level or activity of aprotein that phosphorylates a PAK kinase decreases by at least 5, 10,20, 30, 40, 50, 60, or 80, 90, 95, or 100%.

In a related aspect, the invention features another screening method fordetermining whether a compound is useful for treating, stabilizing, orpreventing cancer in a mammal. This method involves measuring thephosphorylation level of merlin (e.g., phosphorylation of serine 518) ina cell, tissue, or mammal in the presence and absence of the compound.The compound is determined to treat, stabilize, or prevent cancer if thecompound decreases the phosphorylation level of merlin. In someembodiments, the method also includes administering the compound to amammal in need of the treatment (e.g., a mammal with cancer or anincreased risk for cancer). In certain embodiments, the compound is amember of a library of at least 5, 10, 15, 20, 30, 50, or morecompounds, all of which are simultaneously administered to the cell,tissue, or mammal. In various embodiments, the compound is a purified orunpurified synthetic organic molecule, naturally occurring organicmolecule, nucleic acid molecule, PAK kinase antisense nucleic acid ordouble stranded RNA molecule, biosynthetic protein or peptide, naturallyoccurring peptide or protein, PAK kinase antibody, or dominant negativePAK kinase protein (e.g., a mutant or fragment of a PAK kinase). Inother embodiments, the compound is an autoinhibitory region of a PAKkinase, such as a protein that includes or consists of at least 25, 50,75, 100, 125, or 150 contiguous amino acids of residues 82-146 of PAK2or the corresponding region in another PAK kinase (e.g., residues 83-149of PAK1). In certain embodiments, the protein includes at least 25, 50,75, 100, 125, 150, 200, or 300 of the N-terminal amino acids of a PAKkinase. In some embodiments, the compound is staurosporine or an ATPanalog. In other embodiments, the compound is an anti-merlin antibody.Desirably, the amount of phosphorylated merlin that is bound by theantibody is at least 2, 5, 10, or 15-fold greater that the amount ofunphosphorylated merlin that is bound. In desirable embodiments, thecompound decreases the percentage of merlin that is phosphorylation orthe total amount of phosphorylated merlin by at least 5, 10, 20, 30, 40,50, 60, 80, 90, 95, or 100%. Exemplary merlin proteins have an aminoacid sequence that is at least 40, 50, 60, 70, 80, 90, 95, or 100%identical to the sequence of a region of human merlin or the sequence offull-length human merlin (accession number P35240).

The invention also features pharmaceutical compositions for thetreatment or prevention of cancer. In one such aspect, the inventionfeatures a pharmaceutical composition that includes one or morecompounds that inhibit PAK kinase activity and/or merlin phosphorylationin an acceptable vehicle. In some embodiments, the composition containsbetween 10 ng and 10 mg, such as between 0.1 to 1 mg, of the compound.In various embodiments, the compound is a synthetic organic molecule,naturally occurring organic molecule, nucleic acid molecule, PAK kinaseantisense nucleic acid or double stranded RNA molecule, biosyntheticprotein or peptide, naturally occurring peptide or protein, PAK kinaseantibody (e.g., an antibody that specifically binds a PAK kinase such asPAK2), or dominant negative PAK kinase protein (e.g., a mutant orfragment of a PAK kinase). In other embodiments, the compound is anautoinhibitory region of a PAK kinase, such as a protein that includesor consists of at least 25, 50, 75, 100, 125, or 150 contiguous aminoacids of residues 82-146 of PAK2 or the corresponding region in anotherPAK kinase (e.g., residues 83-149 of PAK1). In certain embodiments, theprotein includes at least 25, 50, 75, 100, 125, 150, 200, or 300 of theN-terminal amino acids of a PAK kinase. In some embodiments, thecompound is staurosporine or an ATP analog. In other embodiments, thecompound is an anti-merlin antibody. Desirably, the amount ofphosphorylated merlin that is bound by the antibody is at least 2, 5,10, or 15-fold greater that the amount of unphosphorylated merlin thatis bound. In desirable embodiments, the compound decreases thepercentage of merlin that is phosphorylation, the total amount ofphosphorylated merlin, or an activity of a PAK kinase by at least 5, 10,20, 30, 40, 50, 60, 80, 90, 95, or 100%.

Suitable carriers include, but are not limited to, saline, bufferedsaline, dextrose, water, glycerol, ethanol, and combinations thereof.The composition can be adapted for the mode of administration and can bein the form of, for example, a pill, tablet, capsule, spray, powder, orliquid. In some embodiments, the pharmaceutical composition contains oneor more pharmaceutically acceptable additives suitable for the selectedroute and mode of administration. These compositions may be administeredby, without limitation, any parenteral route including intravenous,intra-arterial, intramuscular, subcutaneous, intradermal,intraperitoneal, intrathecal, as well as topically, orally, and bymucosal routes of delivery such as intranasal, inhalation, rectal,vaginal, buccal, and sublingual. In some embodiments, the pharmaceuticalcompositions of the invention are prepared for administration tovertebrate (e.g., mammalian) subjects in the form of liquids, includingsterile, non-pyrogenic liquids for injection, emulsions, powders,aerosols, tablets, capsules, enteric coated tablets, or suppositories.

Exemplary cancers that can be treated, stabilized, or prevented usingthe above methods include cancers of the nervous system (e.g., Schwanncell tumors or Nf2), prostate cancers, breast cancers, ovarian cancers,pancreatic cancers, gastric cancers, bladder cancers, salivary glandcarcinomas, gastrointestinal cancers, lung cancers, colon cancers,melanomas, brain tumors, leukemias, lymphomas, and carcinomas. Benigntumors may also be treated or prevented using the methods and compoundsof the present invention. Exemplary mammals include humans, primatessuch as monkeys, animals of veterinary interest (e.g., cows, sheep,goats, buffalos, and horses), and domestic pets (e.g., dogs and cats).

With respect to the therapeutic methods of the invention, it is notintended that the administration of compounds to a mammal be limited toa particular mode of administration, dosage, or frequency of dosing; thepresent invention contemplates all modes of administration, includingoral, intraperitoneal, intramuscular, intravenous, intraarticular,intralesional, subcutaneous, or any other route sufficient to provide adose adequate to prevent or treat cancer. One or more compounds may beadministered to the mammal in a single dose or multiple doses. Whenmultiple doses are administered, the doses may be separated from oneanother by, for example, one week, one month, one year, or ten years. Itis to be understood that, for any particular subject, specific dosageregimes should be adjusted over time according to the individual needand the professional judgment of the person administering or supervisingthe administration of the compositions. If desired, conventionaltreatments such as radiation therapy, chemotherapy, and/or surgery maybe used in combination with the compounds of the present invention.

In various embodiments of any of the aspects of the invention, thecompound has a molecular weight contained in one of the followingranges: 100-4,000 daltons, 100-3,000 daltons; 100-2,000 daltons;100-1,000 daltons; 100-750 daltons; 250-4,000 daltons, 250-3,000daltons; 250-2,000 daltons; 250-1,000 daltons; 250-750 daltons;400-4,000 daltons, 400-3,000 daltons; 400-2,000 daltons; 400-1,000daltons; or 400-750 daltons, inclusive.

By “AK kinase” is meant a protein with an amino acid sequence that is atleast 40, 50, 60, 70, 80, 90, 95, or 100% identical to the sequence of aregion (e.g., a region of at least 50, 100, 150, or 200 amino acids) ofa p21-activted kinase (PAK) or the sequence of a full-length PAK.Exemplary PAK kinases have a sequence at least 40, 50, 60, 70, 80, 90,95, or 100% identical to human PAK1, 2, 3, 4, 5, or 6 over its entiresequence. Examples of human PAK kinase sequences are deposited under thefollowing accession numbers: Q13153 (PAK1), Q13177 (PAK2), O75914(PAK3), NP_(—)005875 (PAK4), BAA94194 (PAK5), and NP_(—)064553 (PAK6).Desirably, the level of an activity of the PAK kinase (e.g.,phosphorylation of merlin) is at least 30, 50, 60, 70, 80, 90, 95, or100% of the level of the corresponding activity of human PAK1, 2, 3, 4,5, or 6. PAK kinases belong to a larger group of the STE20-like kinases.

By “compound that decreases PAK kinase activity” is meant a compoundthat decreases the level of a PAK kinase mRNA or protein, an activity ofa PAK kinase, the half-life of a PAK kinase mRNA or protein, or thebinding of a PAK kinase to another molecule (e.g., a substrate for a PAKkinase, a Rac protein, or a cdc42 protein), as measured using standardmethods (see, for example, Ausubel et al., Current Protocols inMolecular Biology, Chapter 9, John Wiley & Sons, New York, 2000). Forexample, the compound may directly or indirectly inhibit the ability ofa PAK kinase to phosphorylate merlin. In other desirable embodiments, acompound that decreases PAK kinase activity reduces or stabilizes thelevel of Rac or cdc42 mRNA or protein and thus reduces or stabilizes thelevel of an activated PAK kinase. mRNA expression levels may bedetermined using standard RNase protection assays or in situhybridization assays, and the level of protein may be determined usingstandard Western or immunohistochemistry analysis (see, for example,Ausubel et al., supra). The phosphorylation levels of signaltransduction proteins downstream of merlin acitivity may also bemeasured using standard assays. Desirably, the compound decreases PAKkinase activity by at least 20, 40, 60, 80, or 90%. In another desirableembodiment, the level of PAK kinase activity is at least 2, 3, 5, 10,20, or 50-fold lower in the presence of the compound. In yet anotherdesirable embodiment, the compound preferentially decreases theexpression or kinase activity of PAK2; for example, the compound maydecrease the expression or kinase activity of PAK2 by at least 50, 100,200 or 500% more than it decreases the expression or kinase activity ofanother PAK kinase, such as PAK1, PAK3, PAK4, PAK5, or PAK6. Otherdesirable compounds decrease the expression or kinase activity ofmultiple PAK kinases (e.g., 2, 3, 4, 5, 6, or more PAK kinases).Desirably, the level of PAK2 mRNA, PAK2 protein, or PAK2 kinase activityin the presence of the compound is less than 80, 60, 40, or 20% of thecorresponding level in the absence of the compound. Desirably, thedecrease in PAK kinase activity in the central nervous system is atleast 2, 3, 5, 10, 20, or 50-fold greater than the decrease in PAKkinase activity in the periphery or than the decrease in the activity ofanother kinase. It is also contemplated that the expression or activityof a protein having an amino acid sequence that is substantiallyidentical to that of a PAK kinase may be inhibited.

Compounds that may be tested for their ability to decrease PAK kinaseactivity include, but are not limited to, synthetic organic molecules,naturally occurring organic molecules, nucleic acid molecules, PAKkinase antisense nucleic acids or double stranded RNA molecules,biosynthetic proteins or peptides, naturally occurring peptides orproteins, PAK kinase antibodies, or dominant negative PAK kinaseproteins. Because sequences within PAK kinases are autoinhibitory,peptides or peptide analogs based on these autoinhibitory regions may beused as inhibitors of PAK kinase activity (Maruta et al., Ann. N.Y.Acad. Sci., 886:48-57, 1999). Additionally, the autoinhibitory domain ofPAK2 that is described herein may be used as an inhibitor of PAK kinaseactivity or may be used as an initial structure for the design of otherpeptides or peptide analogs that inhibit PAK kinase activity. Otherexemplary PAK kinase inhibitors include staurosporine, staurosporineanalogs, and pharmacuetically acceptable salts thereof (Zeng et al., J.Cell Sci. 113 (Pt 3): 471-82, 2000; Yu et al., J Biochem (Tokyo),129(2): 243-51, 2001). Exemplary PAK kinase inhibitors which may inhibitPAK kinases indirectly include those reported by He et al. (Cancer J.7(3): 191-202, 2001, Cancer J. 6(4):243-8, 2000). Still other preferredcompounds include ATP analogs.

By “antibody that specifically binds a protein” is meant an antibodythat binds to a PAK kinase or merlin, but does not substantially bind toother molecules in a sample, e.g., a biological sample, that naturallyincludes a PAK kinase or merlin. Desirably, the amount antibody bound toa PAK kinase or merlin is at least 50%, 100%, 200%, 500%, or 1,000%greater than the amount of antibody bound to other proteins under thesame conditions. In some embodiments, the amount of antibody bound toPAK2 is at least 2, 5, 10, or 20-fold more than the amount bound toanother PAK kinase, such as PAK1, PAK3, PAK4, PAK5, or PAK6. Desirably,the antibody decreases the activity of a PAK kinase and/or thephosphorylation level of merlin by at least 30, 50, 60, 70, 80, 90, 95,or 100%. In various embodiments, the antibody is a modified antibody,bifunctional antibody, or antibody fragment.

By “modified antibody” is meant an antibody having an altered amino acidsequence so that fewer antibodies and/or immune responses are elicitedagainst the modified antibody when it is administered to a mammal suchas a human. For example, the constant region of the antibody may bereplaced with the constant region from a human antibody. For the use ofthe antibody in a mammal other than a human, an antibody may beconverted to that species format.

By “bifunctional antibody” is meant an antibody that includes anantibody or a fragment of an antibody covalently linked to a differentantibody or a different fragment of an antibody. In one preferredembodiment, both antibodies or fragments bind to different epitopesexpressed on a PAK kinase. Other preferred bifunctional antibodies bindto two different antigens, such as to two different PAK kinases.Standard molecular biology techniques such as those described herein maybe used to operably link two nucleic acids so that the fusion nucleicacid encodes a bifunctional antibody.

By “fragment” is meant a polypeptide having a region of consecutiveamino acids that is identical to the corresponding region of an antibodyof the invention but is less than the full-length sequence. The fragmenthas the ability to bind the same antigen as the corresponding antibodybased on standard assays, such as those described herein. Desirably, thebinding of the fragment to a PAK kinase is at least 20, 40, 60, 80, or90% of that of the corresponding antibody.

By “antisense” is meant a nucleic acid, regardless of length, that iscomplementary to the coding strand or mRNA of a PAK kinase. In someembodiments, the antisene molecule inhibits the expression of only onePAK kinase, and in other embodiments, the antisense molecule inhibitsthe expression of more than one PAK kinase. Desirably, the antisensenucleic acid decreases the expression or biological activity of a PAKkinase by at least 20, 40, 50, 60, 70, 80, 90, 95, or 100%. A antisensemolecule can be introduced, e.g., to an individual cell or to wholeanimals, for example, it may be introduced systemically via thebloodstream.

In some embodiments, the antisense molecule is less than 200, 150, 100,75, 50, or 25 nucleotides in length. In other embodiments, the antisensemolecule is less than 50,000; 10,000; 5,000; or 2,000 nucleotides inlength. In certain embodiments, the antisense molecule is at least 200,300, 500, 1000, or 5000 nucleotides in length. In some embodiments, thenumber of nucleotides in the antisense molecule is contained in one ofthe following ranges: 5-15 nucleotides, 16-20 nucleotides, 21-25nucleotides, 26-35 nucleotides, 36-45 nucleotides, 46-60 nucleotides,61-80 nucleotides, 81-100 nucleotides, 101-150 nucleotides, or 151-200nucleotides, inclusive. In addition, the antisense molecule may containa sequence that is less than a full length sequence or may contain afull-length sequence.

By “double stranded RNA” is meant a nucleic acid containing a region oftwo or more nucleotides that are in a double stranded conformation. Invarious embodiments, the double stranded RNA consists entirely ofribonucleotides or consists of a mixture of ribonucleotides anddeoxynucleotides. The double stranded RNA may be a single molecule witha region of self-complimentarity such that nucleotides in one segment ofthe molecule base pair with nucleotides in another segment of themolecule. Alternatively, the double stranded RNA may include twodifferent strands that have a region of complimentarity to each other.Desirably, the regions of complimentarity are at least 70, 80, 90, 95,98, or 100% complimentary. Desirably, the region of the double strandedRNA that is present in a double stranded conformation includes at least5, 10, 20, 30, 50, 75, 100, 200, 500, 1000, 2000 or 5000 nucleotides orincludes all of the nucleotides in the double stranded RNA. Desirabledouble stranded RNA molecules have a strand or region that is at least70, 80, 90, 95, 98, or 100% identical to a coding region or a regulatorysequence (e.g., a transcription factor binding site, a promoter, or a 5′or 3′ untranslated region) of a PAK kinase. In some embodiments, thedouble stranded RNA is less than 200, 150, 100, 75, 50, or 25nucleotides in length. In other embodiments, the double stranded RNA isless than 50,000; 10,000; 5,000; or 2,000 nucleotides in length. Incertain embodiments, the double stranded RNA is at least 200, 300, 500,1000, or 5000 nucleotides in length. In some embodiments, the number ofnucleotides in the double stranded RNA is contained in one of thefollowing ranges: 5-15 nucleotides, 16-20 nucleotides, 21-25nucleotides, 26-35 nucleotides, 36-45 nucleotides, 46-60 nucleotides,61-80 nucleotides, 81-100 nucleotides, 101-150 nucleotides, or 151-200nucleotides, inclusive. In addition, the double stranded RNA may containa sequence that is less than a full-length sequence or may contain afull-length sequence.

In some embodiments, the double stranded RNA molecule inhibits theexpression of only one PAK kinase, and in other embodiments, the doublestranded RNA molecule inhibits the expression of more than one PAKkinase. Desirably, the nucleic acid decreases the expression orbiological activity of a PAK kinase by at least 20, 40, 50, 60, 70, 80,90, 95, or 100%. A double stranded RNA can be introduced, e.g., to anindividual cell or to whole animals, for example, it may be introducedsystemically via the bloodstream.

In various embodiments, the double stranded RNA or antisense moleculeincludes one or more modified nucleotides in which the 2′ position inthe sugar contains a halogen (such as flourine group) or contains analkoxy group (such as a methoxy group) which increases the half-life ofthe double stranded RNA or antisense molecule in vitro or in vivocompared to the corresponding double stranded RNA or antisense moleculein which the corresponding 2′ position contains a hydrogen or anhydroxyl group. In yet other embodiments, the double stranded RNA orantisense molecule includes one or more linkages between adjacentnucleotides other than a naturally-occurring phosphodiester linkage.Examples of such linkages include phosphoramide, phosphorothioate, andphosphorodithioate linkages.

By “purified” is meant separated from other components that naturallyaccompany it. Typically, a factor is substantially pure when it is atleast 50%, by weight, free from proteins, antibodies, andnaturally-occurring organic molecules with which it is naturallyassociated. Desirably, the factor is at least 75%, more desirably, atleast 90%, and most desirably, at least 99%, by weight, pure. Asubstantially pure factor may be obtained by chemical synthesis,separation of the factor from natural sources, or production of thefactor in a recombinant host cell that does not naturally produce thefactor. Proteins, vesicles, organelles, and small molecules may bepurified by one skilled in the art using standard techniques such asthose described by Ausubel et al. (Current Protocols in MolecularBiology, John Wiley & Sons, New York, 2000). The factor is desirably atleast 2, 5, or 10 times as pure as the starting material, as measuredusing polyacrylamide gel electrophoresis, column chromatography, opticaldensity, HPLC analysis, or western analysis (Ausubel et al., supra).Preferred methods of purification include immunoprecipitation, columnchromatography such as immunoaffinity chromatography, magnetic beadimmunoaffinity purification, and panning with a plate-bound antibody.

By “treating, stabilizing, or preventing cancer” is meant causing areduction in the size of a tumor, slowing or preventing an increase inthe size of a tumor, increasing the disease-free survival time betweenthe disappearance of a tumor and its reappearance, preventing an initialor subsequent occurrence of a tumor, or reducing an adverse symptomassociated with a tumor. In one desirable embodiment, the number ofcancerous cells surviving the treatment is at least 20, 40, 60, 80, or100% lower than the initial number of cancerous cells, as measured usingany standard assay. Desirably, the decrease in the number of cancerouscells induced by administration of a compound of the invention is atleast 2, 5, 10, 20, or 50-fold greater than the decrease in the numberof non-cancerous cells. In yet another desirable embodiment, the numberof cancerous cells present after administration of an compound thatinhibits PAK kinase activity or inhibits merlin phosphorylation is atleast 2, 5, 10, 20, or 50-fold lower than the number of cancerous cellspresent prior to the administration of the compound or afteradministration of a buffer control. Desirably, the methods of thepresent invention result in a decrease of 20, 40, 60, 80, or 100% in thesize of a tumor as determined using standard methods. Desirably, atleast 20, 40, 60, 80, 90, or 95% of the treated subjects have a completeremission in which all evidence of the cancer disappears. Desirably, thecancer does not reappear or reappears after at least 5, 10, 15, or 20years. Examples of cancers that may be treated using these methodsinclude familial and sporadic tumors of the nervous system, such asschwannomas, meningiomas, or ependymomas.

By “mutation” is meant an alteration in a naturally-occurring orreference nucleic acid sequence, such as an insertion, deletion,frameshift mutation, silent mutation, nonsense mutation, or missensemutation. Desirably, the amino acid sequence encoded by the nucleic acidsequence has at least one amino acid alteration from anaturally-occurring sequence.

By “substantially identical” is meant having a sequence that is at least60, 70, 80, 90, 95, or 100% identical to that of another sequence.Sequence identity is typically measured using sequence analysis softwarewith the default parameters specified therein (e.g., Sequence AnalysisSoftware Package of the Genetics Computer Group, University of WisconsinBiotechnology Center, 1710 University Avenue, Madison, Wis. 53705). Thissoftware program matches similar sequences by assigning degrees ofhomology to various substitutions, deletions, and other modifications.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C illustrate the result of a kinase assay withmerlin-substrate and full-length merlin. In vitro kinase assay wasperformed with the GST-merlin fragment pseudo-substrate (FIGS. 1A and1B) or with full-length merlin (FIG. 1C). Substrates were precipitatedand resolved by SDS-PAGE and exposed to film. “W.T.” denotes wild-type;“S518A” denotes alanine mutant; “S518T” denotes threonine mutant; “Ind”denotes serum induced extract, and “606 N.I.” denotes non induced.

FIGS. 2A and 2B illustrate the chromatographic separation of merlinkinase activity. Merlin kinase activity was separated on a Q-sepharose10_(—)10 column. Fractions were monitored for activity by in-vitrokinase assay with the pseudo-substrate and Western blot analysis forPAK3 (FIG. 2A). In a subsequent step, fractions 13-15 were furtherresolved on a Matrex Red dye-ligand column. Kinase activity and PAK2were followed (FIG. 2B). “L” denotes the load fraction.

FIGS. 3A-3C illustrate the in-vivo analysis of merlin phosphorylation byPAK2. NIH3T3 cells were transfected with various expression vectors andwere analyzed by western blot 48 hours post transfection. Extractsprepared from cells transfected with wild type merlin or merlin SS518Awith expression vector for active PAK2 (PAK2-^(T402E)) (FIG. 3A).Extracts prepared from cells transfected with Active Rae (Rae L61) oractive cdc42 (cdc42 V12), full length merlin, and the PAK2autoinhibitory domain (AID) (FIG. 3B). Extracts from cells transfectedwith various mutants of Rac or cdc42 and wild-type merlin (FIG. 3C).

FIGS. 4A-4C show the kinetics of merlin phosphorylation and PAKactivation. Western blot analysis of merlin phosphorylation in NIH3T3cells grown in suspension (sus) and 5′, 20′, and 60′ after re-plating ontissue culture plates (FIG. 4A). Typical profile of PAK activation asmeasured by fold phosphorylation of histone H4. Activity was assayed at5′, 10′, 20′, and 60′ after re-plating and compared to cells grown insuspension 0′ (FIG. 4B). NIH3T3 extracts were immunodepeleted with PAK2or PAK3 antibodies and analyzed by western blot. The merlin-kinaseactivity in the treated extracts was determined by an in-vitro kinaseassay with the merlin substrate and resolved by SDS-PAGE and exposed tofilm (FIG. 4C).

FIGS. 5A-5C illustrate the binding of merlin to phospho-serine 518antibodies and the subcellular localization of merlin. Western blotanalysis was performed on wild-type and S518A merlin expressed inLLC-PK1 cells. “SC-331” denotes commercial antibodies, and “HM2175”denotes phosphospecific antibodies. FIG. 5A is a picture of the Westernblot analysis of increasing amounts of merlin precipitated from LLC-PK1extracts. FIG. 5B is a picture of the Western blot analysis of wild-typeand S518A merlin in LLC-PK1 transfected cells. FIG. 5C is a pictureillustrating the immuno-localization of merlin with SC331 and HM2175.Wild type merlin (panel 1), merlin S518A (panel 2), merlin S518D (panel3), merlin+active PAK2 (panel 4), staining with HM2175 of wild typemerlin (panel 5) and merlin S518A (panel 6).

DETAILED DESCRIPTION

We discovered that the phosphorylation of the tumor suppressor merlin atserine 518 is induced by the p21-activated kinase PAK2. Thisphosphorylation of merlin was demonstrated by biochemical fractionation,use of active and dominant-negative mutants of PAK2, effector activationdefective mutants of Rac/cdc42, immunodepletion, andco-immunoprecipitation. Wild-type and mutated forms of merlin andphospho-specific antibodies were used to show that phospho-merlin isenhanced in membrane protrusions in epithlial cells.

The data regarding merlin phosphorylation indicate that phosphorylationof merlin “inactivates” merlin's function by opening the protein anddisrupting merlin intra- and inter-molecular associations. Thus,phosphorylation of merlin by PAK may be equated to inactivation ofmerlin by disease causing mutations. In light of this information, andthe fact that a large number of the above-mentioned tumor types of thecentral nervous system do not present with mutations in the Nf2 locus,we believe that the required inactivation of merlin in the tumors isachieved by up-regulation of merlin phosphorylation by p21-activatedkinases. Hence, administration of a specific p21-activated kinaseinhibitor may down regulate merlin phosphorylation and restore merlinfunction as a tumor suppressor.

Thus, a variety of cancers (e.g., cancers of the central nervous system)can be prevented or treated by administering one or more compounds thatinhibit PAK kinase activity and/or phosphorylation of merlin. Compoundsuseful in these methods can be identified in standard assays such asthose described herein. These therapeutic and screening methods aredescribed further below.

Methods for Analysis of PAK Kinase Activity and Merlin PhosphorylationPlasmids and Transfections

Wild-type merlin and S518A were subcloned from previously describedvectors (Shaw et al., supra) into the BamH1-EcoRI sites of pcDNA3(Invitrogen). The merlin GST-substrates were prepared by PCRamplification of the 60 amino acid coding sequences from the wild-typeand S518A versions of merlin. The primers used were:5′-CCAGGAATTCCATTGCCACCAAGCCCACGTACCCG-3′ (SEQ ID NO:1) and5′-GCAAGCATCTGCAGGAGCACCTCGACTCGAGCGGCC-3′ (SEQ ID NO:2). The fragmentswere then cut with EcoRI and XhoI and ligated into pGEX5-3x. Pak2wild-type and active forms: wild-type PAK2 was amplified and cloned intothe BamHI and XhoI sites of pcDNA3 (Invitrogen). The T402E mutation wascreated by site-directed mutagenesis using the Quick-change kit, asinstructed by the manufacturer (Stratgene). The primers used were:5′-GCAGAGCAAACGCAGTGAGATGGTCGGAACGCC-3′ (SEQ ID NO:3) and5′-GGCGTTCCGACCATCTCACTGCGTTTGCTCTGC-3′ (SEQ ID NO:4). The PAK2 autoinhibitory domain was amplified using5′-CCGCTCGAGATGCACACCATCCATGTTGGC-3′ (SEQ ID NO:5) and5′-GCTCTAGATTAATCTTTCTCAGGAGGAGTAAAGC-3′ (SEQ ID NO:6) and cloned intothe XhoI and XbaI sites of pcDNA3. All plasmids were transfected in tothe various cell lines using Lipofectamine (Life Technologies) accordingto manufacturer's instructions.

In-Vitro Kinase Assay

Total cellular extracts were prepared by lysing the cells directly incell lysis buffer; 50 mm HEPES pH=7.4, 1% NP-40, 150 mM NaCl, 25 mM NaF,20 mM β-glycerophosphate, 1 mM EDTA, and protease inhibitors. Theextracts were added into reaction tubes containing 200 ng GST-substrate,2 mM MgCl, 2 mM DTT, 100M cold ATP and 10 μCi p³² [γ]-ATP. For theimmunodepeletion experiments extracts were incubated with the relevantantibody for three hours at 4° C. After four sequential exchanges ofantibody, the presence of the protein in question was determined bywestern blot analysis. Kinase reactions were carried out at 30° C. for20 minutes. The substrate was washed three times in lysis buffer at 4°C. Termination of the reaction was by addition of protein sample bufferand boiling for five minutes. The samples were then resolved bySDS-PAGE, dried on 3 MM paper (Whatman), and exposed to X-ray film.

Merlin Phosphorylation

In the in-vitro kinase assays where full-length merlin was used, cellstransfected with merlin expression vectors were extracted, and merlinwas immunoprecipitated using a commercially available antibody SC-331(Santa Cruz) and Protein A-agarose beads. In the in-vivo studies, cellswere transfected with various expression vectors and harvested after 48hours into SDS-boiling buffer (10 mM Tris pH 7.5, 50 mM NaF, and 1%SDS). Cells were scraped off the plates and boiled for 5′. Proteinconcentration was determined by the BCA method (Pierce) and resolved by9% SDS-PAGE and western blot analysis.

Kinetics of Merlin Phlosphorylation and PAK Activation

In the time course study of merlin phosphorylation, NIH3T3 cells weretransfected with either merlin or PAK2 expression vectors. Fourty-eighthours post transfection, the cells were serum starved for 24 hours andthen trypsinized, washed, and transferred to poly-HEME coated plates forthree hours. Cells were then plated unto coated tissue culture dishesfor various periods of time and collected into SDS-boiling buffer. Theprotein was concentration was determined, and the protein was resolvedon 9% SDS-PAGE and western blot analysis. For activation of PAK2, theextracts were immunoprecipitated with a commercially available antibody(Zymed) and resolved in an in-gel kinase assay using histone H4, aspreviously described (Price et al., Mol. Biol. Cell 9:1863-1871, 1998).

Production of Phospho-Specific Antibodies and Commercial Antibodies

A chemically phosphorylated peptide: [H}-CKDTDMKRLS*MEIE—[NH2] (SEQ IDNO: 7 was coupled to SulfoLink coupling gel (Pierce) and used toimmunize rabbits. A standard protocol of immunization was employed. Serafrom the animals were then purified in two steps. First the sera waspassed over an affinity column of the phospho-antigen. This column bindsantibodies recognizing the phosphorylated and/or unphosphorylated formsof the peptide. In a second step, the bound fraction of the first columnwas applied to a second column of the unphosphorylated peptide. Theflow-through was collected and contained only phospho-specificantibodies.

Biochemical Fractionation of Merlin-Kinase Activity

Ion-exchange chromatography was performing using a Q-sepharose(Pharmacia) 10_(—)10 column at pH=8.5 (Diethanolamine buffer) to which alinear NaCl gradient was applied. Maximal activity eluted around 120-130mM NaCl. A 60-fold enrichment was achieved with overall recovery ofabout 70%. For dye-ligand chromatography, a Matrex Red A dye (Millipore)10_(—)10 column was used (pH 8, 0-500 mM KCl linear gradient), yieldinga 10-fold enrichment with 25% recovery. For gel filtration, best resultswere achieved with a 10_(—)30 superose 6 column (Pharmacia) at a flowrate of 0.2 ml/min.

Immunoflouresence

NIH3T3 or LLC-PK1 cells were plated on glass cover slips and transfectedwith various expression vectors. Twenty-four hours post transfectioncells were fixed in 4% paraformaldhyde for 15′ and permabilized withTriton X-100 for 10′. Sc-331 was used at 1:1000 dilution and HM2175 wasused at 1.0 μg/ml.

Merlin Pseudo-Substrate is Phosphorylated In-Vitro by PAK2

Merlin is phosphorylated in response to various stimuli such as serumstarvation, confluency and detachment. To identify the kinase thatphosphorylates merlin on serine 518, an in-vitro kinase assay wasestablished. The assay employs a pseudo-substrate comprised of residues478-535 of merlin fused to glutathione-S-transferase (GST). Thepseudo-substrate was produced in bacteria and then used in an in-vitrokinase assay bound to gluthathione-agarose beads. Kinase activity wasdetermined using SDS-PAGE and autoradiography. The specificity of thisassay was demonstrated by the inability of a substrate in which serine518 was mutated to alanine 518 (S518A) to be phosphorylated to anysignificant extent under the conditions tested (FIG. 1A). Thephosphorylation of the pseudo-substrate was then examined using extractsfrom cells grown under conditions that induce merlin phosphorylation invivo. For example, serum shock and reattachment induce strongphosphorylation of merlin in vivo and of the pseudo-substrate in vitro(FIG. 1B left). When the pseudo-substrate was substituted withfull-length wild-type merlin and merlin S518A in the same in vitrokinase assay, a similar pattern of phosphorylation was observed (FIG.1C).

Once validated, the kinase assay was then employed in a candidate-basedscreen for merlin-kinase. In these screens, NIH3T3 cells weretransfected with the expression vectors for various Rac/cdc42 activatedkinases, and the protein extracts were then employed in the kinase assaywith ATP-γ-P³². After several washes, the substrate was resolved bySDS-PAGE and exposed to film. Only extracts from cells transfected withPAK2 and PAK3 were able to effectively phosphorylate thepseudo-substrate in an in-vitro kinase assay (FIG. 1B). Other kinases,which are known effectors of cdc42/Rac, including PAK1, LIM-kinase andJNK, did not induce phosphorylation of merlin in these assays. Similarresults were observed when the in-vitro kinase assays were preformedwith immunoprecipitated full-length merlin. In this case, the levels ofphosphorylating activity from extracts prepared with RacL61 weresignificantly higher than with the PAK2/PAK3 extracts (FIG. 1C).

PAK2 Levels Correlate with Enrichment for the Merlin Kinase Activity

To discriminate between PAK2 and PAK3 as candidate merlin kinases, thekinase activity was purified over a series of chromatographic steps.Fractions were examined for merlin kinase activity using the in vitrokinase assay and for PAK2 and PAK3 levels by western blotting. After aninitial step of ammonium sulfate precipitation, the precipitatecontaining the activity was separated by ion-exchange chromatography ona Q-sepharose column. As shown in FIG. 2A, the maximal activity ofmerlin kinase was found in fractions 20-22 in this separation. PAK2levels also peaked in fractions 20-22, while PAK3 levels peaked aroundfraction 30. Fractions 20-22 were used in a subsequent step ofdye-ligand chromatography. Maximal activity of merlin kinase wasobserved in fractions 18-22. While PAK2 was present in a pattern thefully overlaps with the peak of merlin kinase activity (FIG. 2B), PAK3was no longer evident in these fractions. After four steps ofenrichment, which included ammonium sulfate precipitation, ion-exchange,dye-ligand chromatography, and gel filtration, a more than 1200-foldenrichment of the kinase activity was achieved. When assessing thefractions of the various chromatographic steps by western blot analysis,PAK2 levels consistently paralleled merlin kinase activity (FIG. 2A).

PAK2 Phosphorylates Merlin In-Vivo

To determine whether PAK2 can induce phosphorylation of merlin in vivo,NIH3T3 cells were transfected with expression vectors for one of twodifferent forms of constitutively active-PAK2 and merlin. PAK2^(T402E),an activated mutant similar to PAK1^(T423E) (Manser et al., Mol. CellBiol. 17:1129-1143, 1997) and PAK2^(Δ1-212), an N-terminal truncatedform similar to the caspase-activated PAK2 were used for this analysis(Rudel et al., J. Immunol. 160:7-11, 1998). Extracts were made 48 hourspost transfection and analyzed by western blot analysis. As shown inFIG. 3A, PAK2^(T402E) caused an increase in the slower migrating,hyperphosphorylated form of merlin. In contrast, the mobility of themerlin S518A mutant was not altered in the presence of PAK2^(T402E).Similar results were observed with PAK2^(Δ1-212). Thus, active PAK2induces phosphorylation of merlin in vivo.

The PAK Auto-Inhibitory Domain Inhibition of the Rac-DependentPhosphorylation of Merlin

The N-terminal regulatory domain of the PAKs has been shown to containan auto-inhibitory domain (AID) (Zhao et al., Mol. Cell Biol.18:2153-2163, 1998). The inhibitory fragment in PAK2 resides betweenresidues 82-146 (equivalent to the 83-149 AID of PAK1). Previous studieshave demonstrated that this domain inhibits PAK activity in trans. Tofurther investigate the role of PAK2 in induction of merlinphosphorylation, the ability of the AID to inhibit this activity wasassessed. NIH3T3 cells were co-transfected with expression vectors formerlin, activated Rac, or cdc42 and the PAK2 AID. As shown in FIG. 3B,the inclusion of the PAK2 AID significantly reduced the phosphorylationof merlin. This result is demonstrated by the 3-4-fold decrease in theratio of hyper-phosphorylated to hypo-phosphorylated merlin in cells inwhich the transfection included the AID. This result indicates thatmerlin-kinase activity induced by Rac/cdc42 is sensitive to PAKinhibition.

Impaired Phosphorylation of Merlin by Effector-Activation DefectiveMutants of Rac/cdc42

Merlin is regulated at least in part by phosphorylation, which isinduced by Rac/cdc42 proteins but not by activated Rho. The Rac andcdc42 proteins belong to the family of the Rho G-proteins. Theseproteins act as molecular switches, cycling between GTP-bound (ON) andGDP-bound (OFF) states. The G-proteins have intrinsic GTPase activitywhich hydrolyzes GTP to GDP. Although they are implicated in theregulation of many signaling pathways, they are mostly associated withregulation of cytoskeleton reorganization, gene expression, and membranetrafficking processes such as endocytosis.

Mutated forms of activated Rac and cdc42 were tested to determine ifthey maintained the ability to induce the phosphorylation of serine 518.The mutants employed were RacV12V37 and cdc42V12A37, which can activatePAKs but are defective in activation of POR1 and ROKa and induction ofcytoskeletal changes, and RacV12K40, which is defective in theactivation of PAKs and JNK but able to activate POR and ROKa and inducecytoskeletal changes (Lamarche et al., Cell 87:519-529, 1996; Joneson etal., Science 274:1374-1376, 1996; and Westwick et al., Mol. Cell Biol.17:1324-1335, 1997). NIH3T3 cells were co-transfected with wild-typemerlin and cdc42V12A37, RacV12V37, or RacV12K40. As shown in FIG. 3C,none of the effector domain mutants induced the phosphorylation ofmerlin to a significant level. These data are consistent with thepossibility that more than one Rac-induced effector is required toinduce full phosphorylation of merlin. In assays using full-lengthmerlin, RacL61 or cdc42V12 always caused a higher degree ofphosphorylation than active forms of PAK2, further suggesting thatadditional Rac/cdc42-initiated events are involved in this process.

Similar Kinetics for the Activation of PAK and Merlin PhosphorylationUpon Re-Attachment of Cell to Substratum

Merlin phosphorylation is reduced when cells are grown in suspension andinduced once the cells are re-plated and begin adhering to thesubstratum (Shaw et al., J. Biol. Chem. 273:7757-7764, 1998). Similarly,PAK activity was shown to be increased upon cell attachment (del Pozo etal., Embo 19:2008-2014, 2000). To assess the kinetics of merlinphosphorylation and PAK2 activation in the present system, NIH3T3 cellswere co-transfected with wild-type PAK2 or merlin, resuspended, and thenallowed to attach to culture plates. Merlin was predominantly in anunphosphorylated state when cells were in suspension (FIG. 4A). However,upon attachment, a steady increase in overall levels of merlin wasdetected up to 20 minutes after plating, then the levels plateaued. Inaddition, a 2-3 fold increase in the levels of the phosphorylated formof merlin was observed five minutes after plating. This level ofphosphorylation remained constant up to one hour after re-plating.

The kinase activity of PAK2 was determined over this time course usingimmunoprecipitated protein and histone H4 as a substrate. As shown inFIG. 4B, PAK2 kinase activity was increased approximately 3-fold at fiveminutes post-attachment. Maximal activity was observed at 20 minutes,and at one hour the activity was reduced again. Thus, thephosphorylation of merlin five minutes after replating correlates wellwith activation of PAK2 kinase activity. The addition of serum to thecells in suspension does not induce activation of PAK2 or thephosphorylation of merlin. This result is in agreement with previousreports and demonstrates the requirement of cell adhesion for bothprocesses.

Reduced Merlin-Kinase Activity Following Immunodepletion of PAK2

To investigate further whether PAK2 is directly responsible for merlinphosphorylation, the enzyme was immunodepleted from extracts ofserum-treated NIH3T3 cells. After four sequential rounds ofimmunoprecipitation, the amount of PAK2 in the extract was reduced, onaverage, by 2-3 fold (FIG. 4C). Treated extracts were then used inkinase assays with the GST-merlin substrate, and activity was determinedusing SDS-PAGE and autoradiography. As shown in FIG. 4C, extractsimmunodepleted for PAK2 (+PAK2 Ab) had a 2-3 fold reduction in kinaseactivity compared to those treated in a similar fashion with ananti-PAK3 antibody (+PAK3 Ab).

Generation of Merlin Phospho-Specific Antibodies

In order to follow the function and localization of merlinphosphorylated at serine 518, antibodies that preferentially recognizethe phosphorylated form (HM2175) were produced by employing achemically-phosphorylated peptide and affinity purification as describedherein. To assess the activity of this antisera, ectopically expressedwild-type merlin and merlin S518A were immunoprecipitated with anonspecific merlin antibody (SC331) and then immunoblotted with eitherSC331 or HM2175. The SC331 antibody detected both the hypo- andhyperphosphorylated form of merlin, while the HM2175 antibodypreferentially bound the hyper-phosphorylated form of the protein (FIG.5A). At high concentrations of merlin, HM2175 did recognize thehypophosphorylated form to some extent. When merlin S518A was tested ina similar approach, HM2175 did not recognize the protein, even whenloaded at high concentrations (FIG. 5B).

The specificity of the phospho-directed antibodies was tested usingimmunocytochemistry. LLC-PK1 cells were transfected with vectorsexpressing either wild-type merlin or the merlin S518A mutant. Fixedcells were then incubated with either the SC331 or HM2175 antibodies.The SC331 antibody recognized both wild-type and mutant merlin (FIG. 5Cpanels 1 and 2); in contrast, the HM2175 antibody recognized onlyectopic wild-type merlin (FIG. 5B panels 5 and 6). This pattern ofspecificity was observed in many cell lines of both epithelial andfibroblastic origin.

Phosphorylation Leads to Changes in the Subcellular Localization ofMerlin in LLC-PK1 Cells

To visualize subcellular localization of the different forms of merlin,LLC-PK1 cells were transfected with wild-type merlin or the merlin S518Aor S518D mutants and incubated with the SC331 antibody. As shown in FIG.5B (panel 1), wild-type merlin mainly localized to microvilli. Loweramounts of merlin were present in larger membrane protrusions and incortical actin structures. Similar results were obtained with the S518Amutant (panel 2). However, the pseudo-phosphorylated mutant of merlin(S518D) was localized predominantly in the larger membrane protrusions(panel 3). This localization was also observed when wild-type merlin wasco-transfected with active PAK2 to increase merlin phosphorylation(panel 4). Importantly, the localization of the S518A mutant was notaffected by co-transfected PAK2. To validate the observation that merlinrelocalization is a result of serine 518 phosphorylation, HM2175 wasused to stain cells for phosphorylated merlin. As expected, the HM2175antibody stained wild-type merlin mainly in the cellular protrusions(panel 5) but did not stain the merlin S518A mutant (panel 6). Thisresult reinforces the conclusion that the phosphorylation of serine 518is required for the redistribution of merlin and that the redistributionof the merlin 5518D mutant was not simply the consequence of mutatingserine 518 to aspartic acid.

Thus, wild-type merlin and the S518A mutant were mostly localized tomicrovilli and to a small number of protrusions and cortical structuresin LLC-PK1 cells. However, when the merlin S518D mutant or wild-typemerlin plus PAK2^(−T402E) were introduced into these cells, a markedredistribution of merlin to membrane protrusions was observed. Using theHM2175 antibody to stain both wild-type merlin alone or plusPAK2^(−402E) produced results similar to those obtained using the SC331antibody. Other modified forms of merlin could not be observed with thisantibody. Merlin is not exclusively localized to the protrusions: merlincan be observed in microvilli and cortical actin at a somewhat reducedlevel. The redistribution is dependent on phosphorylation of serine 518;for example, when merlin S518A was transfected with PAK₂ ^(−T402E) theshift in localization was not observed. This redistribution of merlin ishighly reminiscent of the redistribution of Ezrin in response tophosphorylation.

Induction of Merlin Phosphorylation by PAK2

Several independent lines of evidence are presented herein whichimplicate PAK2 as the kinase responsible for inducing phosphorylation ofserine 518 on the Nf2 tumor suppressor gene product, merlin. Only PAK2and PAK3 induced the phosphorylation of the pseudo-substrate in-vitroand the full-length merlin in vivo. We were able to separate PAK2 fromPAK3 and demonstrate that merlin-kinase activity migrates in parallel toPAK2. The use of the active PAK2, the dominant negative fragmentPAK2-AID, and immunodepeletion of PAK2 directly reinforce theidentification of PAK2 as the merlin kinase. Additional circumstantialevidence includes similar kinetics and conditions for PAK2 activationand merlin phosphorylation. The serine 518 site of phosphorylation issimilar to many PAK recognition sites such as the phox p47 site (RKRLSQ,SEQ ID NO: 8 vs. MKRLSM, SEQ ID NO: 9) which has been shown to be an invivo substrate of the PAKs (Knaus et al., Science 269:221-223, 1995 andDing et al., J. Biol. Chem. 271:24869-24873, 1996). Finally, variouskinase inhibitors were tested for their ability to inhibit the activityof the merlin kinase. Staurosporine inhibited the merlin kinase atconcentrations in the lower nM range. The inhibitors K252a and K-5720were inhibitory in the mM range, while H9 had no effect up to aconcentration of 1 mM. This inhibition spectrum is in close agreementwith previous data regarding PAK inhibition (Zeng et al., J. Cell Sci.113:471-482,2000 and Yu et al., J. Biochem. (Tokyo) 129:243-251, 2001).

The results of the transfection of cells with mutated forms of Racreinforces the role of PAK2 in merlin phosphorylation and raises thepossibility that additional mechanisms are involved in the regulation ofmerlin. RacV12K40 and RacV12V37 are defective in signaling towardscytoskeleton and transcriptional events, respectively. In contrast,RacV12K40 activates PAKs but does not induce JNK activation; however, itactivates both ROK and POR1, leading to membrane ruffling. In contrast,RacV12V37 activates PAK but does not activate ROK or POR1, thus inducingJNK activation but no membrane ruffling. When cells were transfectedwith these mutated forms of Rac, merlin was not phosphorylated abovebasal levels. The fact that RacV12K40 does not induce phosphorylation ofmerlin further supports the role of PAK2 in the phosphorylation ofmerlin (Lamarche et al., supra; Joneson et al., supra, and Westwick etal., supra). However, RacV1K37, which can activate PAK, does not inducethe phosphorylation of merlin either. This result is consistent with thepossibility of multiple Rac-induced pathways for the induction of merlinphosphorylation. This result may also correlate with the inability ofthe PAK-AID to fully inhibit merlin phosphorylation. As demonstrated forERMs, multiple effectors can play a role in conformational regulationincluding phosphorylation and phospholipids (Hirao et al., J. Cell Biol.135:37-51, 1996 and Matsui et al., supra). Thus, similar multipleeffectors may function in the regulation of merlin. The binding ofmerlin to CD44 has been shown to be enhanced by including phospholipidsin the reaction, perhaps by “opening” the structure of merlin andrevealing the CD44 interacting domain (Sainio et al., J. Cell Sci.110:2249-2260, 1997). Thus, phosphorylation of serine 518 andphospholipids may contribute, concomitantly or sequentially, to the“opening” of merlin. Compared to the efficiency of phosphorylation offull-length merlin, the pseudo-substrate is phosphorylated at a muchhigher efficiency. This result could be due to the fact that thepseudo-substrate, being out of the context of the full-length protein,is more accessible to PAK2. Similar phenomena have been reported inprevious studies looking at the phosphorylation of moesin by Rho-kinase.If desired, the effects of phospholipids on the phosphorylation ofmerlin can also be examined.

Direct Interaction Between Merlin and PAK

To confirm the functional interaction between merlin and PAK, twodifferent approaches were employed. First, an interaction between merlinand PAK was demonstrated by co-immunoprecipitation from various celltypes. NIH3T3 cells were transfected with expression vectors for merlinand one of either PAK1, 2 or 3. Protein extracts were prepared andimmunoprecipitated with antibodies against merlin or one of the relevantPAKs. Analysis of the precipitates demonstrated that merlin and eitherPAK1, 2, or 3 can co-immunoprecipitate. In RT4 cells, a similar resultwas obtained using antibodies to precipitate endogenous PAK1 or merlin,indicating that these proteins interact.

To examine whether merlin could inhibit PAK activity, thephosphorylation state of merlin was examined under high or low levels ofmerlin expression. The auto or trans-phosphorylation of PAK1 on serine144 and serine 423 have been demonstrated as significantly contributingto the activation of PAK1. Hence, detection of an increase in levels ofphopspho-PAK1 indicates PAK1 activation (Chong et al., J. Biol. Chem.276:17347-17353, 2001). Thus, the phosphorylation state of PAK1 wasexamined in a Rat schwannoma (RT4-D6P2T) cell line harboring aninducible allele of merlin. The RT4D6P2T cell line is derived from theethylnitrosourea-induced tumor line D6 of the rat peripheral nervoussystem (Tomozawa Sueoka, Proc Natl Acad Sci, USA 75:6305, 1978). Highbasal levels of PAK activation are observed in this cell line. Thephosphorylation status of PAK1 was examined using standard 2-dimensionalprotein analysis under conditions of either high or low merlinexpression. The basal levels of activated PAK1 were reduced underconditions of high merlin expression compared to conditions of lowmerlin expression. Thus, merlin reduces the activation of PAK1 in theRT4 cell line.

Based on the physical association between merlin and PAKs and theability of merlin to inhibit the activation of PAK1, merlin may act as adirect inhibitor of PAKs. Thus, the loss of merlin results in relief ofan inhibition on PAK. This effect may result in unregulated activationof PAK, leading to deleterious effects. This mechanism reinforces theusefulness of inhibitors of PAKs as therapeutic agents for the treatmentor prevention of cancer, such as Nf2 and other disease in which the Nf2gene is impaired.

Mechanisms Involving in Regulation of Merlin

The overall picture emerging from these studies is a mechanisticallysimilar approach of regulation between merlin and the ERMs. Theseproteins are each regulated by multiple mechanisms includingphosphorylation and involvement of phospholipids. In both cases, thesesignals effect the properties of the proteins, including subcellularlocalization and association with the actin cytoskeleton. There are,however, some major differences between merlin and the ERMs with respectto phosphorylation. First, merlin phosphorylation is induced by theRac/cdc42 pathway, while ERM phosphorylation seems to be induced in aRho-dependent fashion. Although there is an extensive network of crosstalk among the Rho family of proteins, some functions remain unique tothe individual members. The study of the Rho family has focused mainlyon their role in regulation of actin dynamics, in which the differentfamily members have different effects (Bishop et al., supra). Thesedifferential effects are mediated by separate effectors that interactwith the small G-proteins. In the case of merlin, PAK2 is involved; forthe ERMs, Rho-kinase and/or PKC-Θ are involved (Pietromonaco et al., J.Biol. Chem. 273:7594-7603, 1998).

Second, the phosphorylation of merlin may render it “inactive” in itsgrowth suppressive role. This hypothesis is supported by experiments inwhich the head-to-tail association of merlin under phosphorylatingconditions was examined. The data suggests that phosphorylation ofmerlin on serine 518 disrupts merlin self-association (Shaw et al.,Developmental Cell 1:63-72, 2001). Taken together with thecircumstantial evidence of merlin phosphorylation under growthpermissive conditions (Shaw et al., 1998 supra), it is possible thatphosphorylation “inactivates” merlin function by opening the protein anddisrupting merlin intra- and inter-molecular associations. Additionally,merlin is found in an hypophosphorylated form when the combination ofcellular and environmental conditions are growth inhibitory. Thisinactivation of merlin by phosphorylation is in contrast to the ERMs,where the opening of the proteins is thought to activate the proteins byrevealing the actin binding and other protein interaction domains(Bretscher et al., Annu. Rev. Cell Dev. Biol. 16:113-143, 2000 andPearson et al., Cell 101:259-270, 2000). This difference may or may notbe related to the observed difference in association of merlin and theERMs with the cytoskeleton. While phosphorylation of merlin at serine518 is required for its dissociation from the cytoskeleton, the oppositeis true for ezrin. Phosphorylation of T567 activates ezrin and enhancesits association with the cytoskeleton; in contrast, a ezrin T567A mutantis poorly associated (Gautreau et al., surpa).

The fact that immunodepeletion significantly reduces phosphorylation ofserine 518 in the in vitro kinase assay implicates PAK2 as directlyphosphorylating merlin. The finding that merlin can inhibit Rac inducedsignaling and that the immediate downstream effector, PAK2,phosphorylates and, perhaps, inactivates merlin reinforces thepossibility of the “positive-feedback” mechanism. In such a model, thesteady-state role of merlin is to down regulate Rac/cdc41-inducedsignaling. Once activated, Rac/cdc42 activate PAK2 which in turnphosphorylates merlin and thus relives its inhibitory effect. From afunctional point of view, this could be achieved by different mechanismsand impinge of these pathways at different levels (Shaw et al.,Developmental Cell 1:63-72, 2001). Merlin could inhibit signaling byacting upstream, by acting upon effectors downstream to Rac/cdc42, oreven by both mechanisms. This possibility would not be unprecedented; ithas been suggested that activation of ERMs is required for theactivation of Rho by LPA and that Rho, in turn, induces phosphorylationof the ERMs (Lamb et al., Nature Cell Biology 2:281-287, 2000). In lightof the present data regarding a shift in merlin localization afterphosphorylation of serine 518, a model in which merlin controls thesubcellular localization of a molecule such as RhoGDI is possible.

It is quite possible that the Rac/cdc42-signaling inhibitory function ofmerlin is also the tumor suppressor function of merlin, as Rac signalingis necessary for transformation. Many examples exist and are reviewed byZohn et al. (Oncogene 17:1415-1438, 1998). Rac activation is requiredfor the full transformed phenotype induced by Tiam and Ras (Habets etal., Cell 77:537-749, 1994; Khosravi-Far et al., Mol. Cell Biol.11:6443-6453, 1995; Qiu et al., Nature 374:457-459, 1995; van Leeuwen etal., Oncogene 11:2215-2221, 1995). Rac has also been shown to regulatecell motility and invasiveness (reviewed by Evers et al., Eur. J. Cancer36:1269-1274, 2000). Examples include the increased metastatic potentialof cells expressing activated Rac (del Posos et al., Oncogene15:3047-3057, 1997) and the identification of the Rac-GEF, Tiam, as apromoter of invasiveness (Habets et al., supra; Keely et al., Nature390:632-636, 1997; Shaw et al., Cell 91:949-960, 1997). The fact thatNf2+/−mice have highly metastatic tumors which have a LOH of thewild-type Nf2 allele agrees with the above mentioned possibilities (Ipand Davis, Curr. Opin. Cell Biol. 10:205-219, 1998).

The identification of an established effector of the Rac/cdc42 pathwaysas the kinase involved in merlin regulation and localization is a stronglink between a well-established signaling pathway and a tumor suppressorgene of unknown function. Thus, the Rac/cdc42 pathways play a role inthe tumor phenotypes in which the Nf2 gene is involved.

Other Compounds for Inclusion in Individual or Combination Therapies

A variety of compounds may be tested for their ability to decrease PAKkinase activity and/or merlin phosphorylation, such as synthetic organicmolecules, naturally occurring organic molecules, nucleic acidmolecules, PAK kinase antisense nucleic acids or double stranded RNAmolecules, biosynthetic proteins or peptides, naturally occurringpeptides or proteins, PAK kinase antibodies, or dominant negative PAKkinase proteins. Additionally, peptides or peptide analogs based on theautoinhibitory regions of PAKs may be used as inhibitors of PAK kinaseactivity (Maruta et al., Ann. N.Y. Acad. Sci., 886:48-57, 1999). ATPanalogs can also be tested for inhibitor activity.

In general, additional drugs for the treatment of cancer may beidentified from large libraries of both natural product or synthetic (orsemi-synthetic) extracts or chemical libraries according to methodsknown in the art. Those skilled in the field or drug discovery anddevelopment will understand that the precise source of test extracts orcompounds is not critical to the methods of the invention.

Accordingly, virtually any number of chemical extracts or compounds canbe screened for their effect on PAK kinase activity and/or merlinphosphorylation. Examples of such extracts or compounds include, but arenot limited to, plant-, fungal-, prokaryotic- or animal-based extracts,fermentation broths, and synthetic compounds, as well as modification ofexisting compounds. Numerous methods are also available for generatingrandom or directed synthesis (e.g., semi-synthesis or total synthesis)of any number of chemical compounds, including, but not limited to,saccharide-, lipid-, peptide-, and nucleic acid-based compounds.Synthetic compound libraries are commercially available from BrandonAssociates (Merrimack, N.H.) and Aldrich Chemical (Milwaukee, Wis.).Alternatively, libraries of natural compounds in the form of bacterial,fungal, plant, and animal extracts are commercially available from anumber of sources, including Biotics (Sussex, UK), Xenova (Slough, UK),Harbor Branch Oceangraphics Institute (Ft. Pierce, Fla.), and PharmaMar,U.S.A. (Cambridge, Mass.). In addition, natural and syntheticallyproduced libraries are produced, if desired, according to methods knownin the art, e.g., by standard extraction and fractionation methods.Furthermore, if desired, any library or compound is readily modifiedusing standard chemical, physical, or biochemical methods.

When a crude extract is found to inhibit PAK kinase activity orphosphorylation of merlin, further fractionation of the positive leadextract is necessary to isolate chemical constituent responsible for theobserved effect. Thus, the goal of the extraction, fractionation, andpurification process is the careful characterization and identificationof a chemical entity within the crude extract. Methods of fractionationand purification of such heterogeneous extracts are known in the art. Ifdesired, compounds shown to be useful agents for the treatment of cancerare chemically modified according to methods known in the art. Compoundsidentified as being of therapeutic value are subsequently analyzed usingany standard animal model of cancer known in the art.

Production of Anti-PAK kinase Antibodies for Treating or PreventingCancer

Anti-PAK kinase antibodies represent exemplary PAK kinase inhibitiorsfor use in the present invention. Anti-PAK kinase antibodies can begenerated using standard methods, such as those described herein. Ifdesired, the ability of an anti-PAK kinase antibody to inhibit PAKkinase activity can be confirmed before the antibody is administered tomammals (e.g., humans) for the treatment or prevention of cancer. Forexample, standard methods can be used to determine the ability ofanti-PAK kinase antibodies to decrease the level of PAK kinase protein,an activity of PAK kinase (e.g., phosphorylation of merlin), thehalf-life of PAK kinase protein, or the binding of PAK kinase to anothermolecule. Anti-PAK kinase antibodies can also be tested in animal orprimate models, such as those described herein, to measure their effecton cancer in vivo.

For the preparation of polyclonal antibodies reactive with PAK kinasefor the treatment or prevention of cancer, one or more PAK kinaseproteins, fragments of PAK kinase, or fusion proteins containing definedportions of PAK kinase can be purified from natural sources (e.g.,cultures of cells expressing PAK kinase) or synthesized in, e.g.,mammalian, insect, or bacterial cells by expression of corresponding DNAsequences contained in a suitable cloning vehicle. Fusion proteins arecommonly used as a source of antigen for producing antibodies. Theantigenic proteins can be optionally purified, and then coupled to acarrier protein, mixed with Freund's adjuvant to enhance stimulation ofthe antigenic response in an inoculated animal, and injected intorabbits, mice, or other laboratory animals. Primary immunizations arecarried out with Freund's complete adjuvant and subsequent immunizationsperformed with Freund's incomplete adjuvant. Following boosterinjections at bi-weekly intervals, the inoculated animals are then bledand the sera isolated. The sera is used directly or is purified prior touse by various methods, including affinity chromatography employingreagents such as Protein A-Sepharose, antigen-Sepharose, andanti-horse-Ig-Sepharose. Antibody titers can be monitored by Westernblot and immunoprecipitation analyses using one or more PAK kinases.Immune sera can be affinity purified using PAK kinase coupled to beads.Antiserum specificity can be determined using a panel of proteins, suchas PAK kinases and other kinases.

Alternatively, monoclonal antibodies are produced by removing the spleenfrom the inoculated animal, homogenizing the spleen tissue, andsuspending the spleen cells suspended in phosphate buffered saline(PBS). The spleen cells serve as a source of lymphocytes, some of whichproduce antibody of the appropriate specificity. These cells are thenfused with permanently growing myeloma partner cells, and the productsof the fusion plated into a number of tissue culture wells in thepresence of selective agents, such as hypoxanthine, aminopterine, andthymidine (Mocikat, J. Immunol. Methods 225:185-189, 1999; Jonak et al.,Hum. Antibodies Hybridomas 3:177-185, 1992; Srikumaran et al., Science220:522, 1983). The wells can then be screened by ELISA to identifythose containing cells making antibody capable of binding to a PAKkinase, fragments, or mutants thereof. These cells can then be re-platedand, after a period of growth, the wells containing these cells can bescreened again to identify antibody-producing cells. Several cloningprocedures can be carried out until over 90% of the wells contain singleclones that are positive for specific antibody production. From thisprocedure, a stable line of clones that produce the antibody areestablished. The monoclonal antibody can then be purified by affinitychromatography using Protein A Sepharose and ion-exchangechromatography, as well as variations and combinations of thesetechniques. Once produced, monoclonal antibodies are also tested forspecific PAK kinase recognition by ELISA, Western blot, and/orimmunoprecipitation analysis (see, e.g., Kohler et al., Nature 256:495,1975; Kohler et al., European Journal of Immunology 6:511, 1976; Kohleret al., European Journal of Immunology 6:292, 1976; Hammerling et al.,In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, New York,N.Y., 1981; Ausubel et al., supra).

As an alternate or adjunct immunogen to a PAK kinase, peptidescorresponding to relatively unique hydrophilic regions of a PAK kinasecan be generated and coupled to keyhole limpet hemocyanin (KLH) throughan introduced C-terminal lysine. Antiserum to each of these peptides canbe similarly affinity-purified on peptides conjugated to BSA, andspecificity tested by ELISA and Western blotting using peptideconjugates, and by Western blotting and immunoprecipitation using a PAKkinase.

Antibodies of the invention are desirably produced using PAK kinaseamino acid sequences that do not reside within highly conserved regions,and that appear likely to be antigenic, as evaluated by criteria such asthose provided by the Peptide Structure Program (Genetics Computer GroupSequence Analysis Package, Program Manual for the GCG Package, Version7, 1991) using the algorithm of Jameson et al., CABIOS 4:181, 1988.These fragments can be generated by standard techniques, e.g., by PCR,and cloned into any appropriate expression vector. For example, GSTfusion proteins can be expressed in E. coli and purified using aglutathione-agarose affinity matrix (Ausubel et al., supra). To minimizethe potential for obtaining antisera that is non-specific or exhibitslow-affinity binding to PAK kinase, two or three fusions may begenerated for each fragment injected into a separate animal. Antiseraare raised by injections in series, preferably including at least threebooster injections.

In addition to intact monoclonal and polyclonal anti-PAK kinaseantibodies, various genetically engineered antibodies and antibodyfragments (e.g., F(ab′)2, Fab′, Fab, Fv, and sFv fragments) can beproduced using standard methods. Truncated versions of monoclonalantibodies, for example, can be produced by recombinant methods in whichplasmids are generated that express the desired monoclonal antibodyfragment(s) in a suitable host. Ladner (U.S. Pat. Nos. 4,946,778 and4,704,692) describes methods for preparing single polypeptide chainantibodies. Ward et al., Nature 341:544-546, 1989, describes thepreparation of heavy chain variable domain which have highantigen-binding affinities. McCafferty et al. (Nature 348:552-554, 1990)show that complete antibody V domains can be displayed on the surface offd bacteriophage, that the phage bind specifically to antigen, and thatrare phage (one in a million) can be isolated after affinitychromatography. Boss et al. (U.S. Pat. No. 4,816,397) describes variousmethods for producing immunoglobulins, and immunologically functionalfragments thereof, that include at least the variable domains of theheavy and light chains in a single host cell. Cabilly et al. (U.S. Pat.No. 4,816,567) describes methods for preparing chimeric antibodies. Inaddition, the antibodies can be coupled to compounds, such as toxins orradiolabels.

Assays and Animal Models for Identifying or Testing Compounds of theInvention

Standard kinase assays, such as the in vitro or in vivo PAK kinaseassays described herein, can be performed in the presence and absence ofone or more candidate compounds to identify compounds that inhibit a PAKkinase (e.g., PAK2). In some embodiments, one or more candidatecompounds are administered to a cell, tissue, or animal that has reducedor negligible levels of merlin (Giovannini et al., Genes Dev.14(13):1617-30, 2000). The reduced levels of merlin may result in ahigher initial level of PAK kinase activity and thus facilitate thedetection of an inhibition of PAK kinase activity by a candidatecompound. Candidate compounds can also be administered to an in vitrosample, cell, tissue, or animal to determine their effect on thephosphorylation of full-length merlin, fragments of merlin, or merlinfusion proteins using standard methods, such as those described herein.

If desired, the compounds of the invention can also be tested for theireffect on cancer using standard animal models for neurofibromatosis type2 (Giovannini et al., supra; McClatchey and Cichowski, Biochim BiophysActa 1471(2):M73-80, 2001). Additionally, animal models for any othertype of cancer (e.g., SCID mouse models) can be used to determine theefficacy of particular compounds or combinations of compounds fortreating or preventing cancer.

Administration of Compunds

A compound of the invention may be administered to humans, domesticpets, livestock, or other animals with a pharmaceutically acceptablediluent, carrier, or excipient, in unit dosage form.

The compounds optionally may be administered as pharmaceuticallyacceptable salts, such as non-toxic acid addition salts or metalcomplexes that are commonly used in the pharmaceutical industry.Examples of acid addition salts include organic acids such as acetic,lactic, pamoic, maleic, citric, malic, ascorbic, succinic, benzoic,palmitic, suberic, salicylic, tartaric, methanesulfonic,toluenesulfonic, or trifluoroacetic acids or the like; polymeric acidssuch as tannic acid, carboxymethyl cellulose, or the like; and inorganicacid such as hydrochloric acid, hydrobromic acid, sulfuric acidphosphoric acid, or the like. Metal complexes include zinc, iron, andthe like.

The chemical compounds for use in such therapies may be produced andisolated as described herein or by any standard technique known to thosein the field of medicinal chemistry. Conventional pharmaceuticalpractice may be employed to provide suitable formulations orcompositions to administer the identified compound to patients sufferingfrom cancer or at increased risk for cancer. Administration may beginbefore or after the patient is symptomatic.

Any appropriate route of administration may be employed. For example,the therapy may be administered either directly to the tumor (forexample, by injection) or systemically (for example, by any conventionaladministration technique). Administration of the compounds may also beoral, topical parenteral, intravenous, intraarterial, subcutaneous,intramuscular, intracranial, intraorbital, ophthalmalic,intraventricular, intracapsular, intraspinal, intracistemal,intraperitoneal, or intranasal. Alternatively, the compounds may beadministered as part of a suppository. Therapeutic formulations may bein the form of liquid solutions or suspensions; for oral administration,formulations may be in the form of tablets or capsules; and forintranasal formulations, in the form of powders, nasal drops, oraerosols. The compounds in a combination therapy may be administeredsimultaneously or sequentially. The dosage of the therapeutic compoundsin a pharmaceutically acceptable formulation depends on a number offactors, including the size and health of the individual patient. Thedosage to deliver may be determined by one skilled in the art.

Methods well known in the art for making formulations are found, forexample, in “Remington: The Science and Practice of Pharmacy” ((19thed.) ed. A. R. Gennaro A R., 1995, Mack Publishing Company, Easton,Pa.). Formulations for parenteral administration may, for example,contain excipients, sterile water, saline, polyalkylene glycols such aspolyethylene glycol, oils of vegetable origin, or hydrogenatednapthalenes. Biocompatible, biodegradable lactide polymer,lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylenecopolymers may be used to control the release of the compounds. Otherpotentially useful parenteral delivery systems include ethylene-vinylacetate copolymer particles, osmotic pumps, implantable infusionsystems, and liposomes. Formulations for inhalation may containexcipients, for example, lactose, or may be aqueous solutionscontaining, for example, polyoxyethylene-9-lauryl ether, glycocholateand deoxycholate, or may be oily solutions for administration in theform of nasal drops, or as a gel.

If desired, treatment with a compound identified according to themethods described above may be combined with more traditional therapiesfor cancer (e.g., cytotoxic agents, radiation therapy, or surgicalremoval of cancerous cells).

Other Embodiments

From the foregoing description, it will be apparent that variations andmodifications may be made to the invention described herein to adopt itto various usages and conditions.

All publications mentioned in this specification are herein incorporatedby reference to the same extent as if each independent publication,patent application, or patent was specifically and individuallyindicated to be incorporated by reference.

1. A screening method for determining whether a compound may be usefulfor treating or stabilizing cancer in a mammal, said method comprisingmeasuring PAK kinase activity in the presence and absence of saidcompound, wherein a decrease in PAK kinase activity is indicative thatsaid compound may be useful to treat or stabilize cancer, wherein saidPAK kinase is a polypeptide comprising a sequence at least 90% identicalto human PAK2, 3, 4, 5, or 6, and wherein said decrease in PAK kinaseactivity is determined by identifying decreased phosphorylation ofmerlin.
 2. A screening method for determining whether a compound may beuseful for treating or stabilizing cancer in a mammal, said methodcomprising measuring the phosphorylation level of merlin in a cell inthe presence and absence of the compound, wherein a decreasedphosphorylation of merlin is indicative that said compound may be usefulto treat or stabilize cancer.
 3. The method of claim 2, wherein saidcompound decreases the percentage of merlin that is phosphorylated orthe total amount of phosphorylated merlin by at least 50%.
 4. The methodof claim 1,wherein said compound is a member of a library of at least 5compounds, all of which are simultaneously administered to a cell. 5.The method of claim 1 or 2, wherein said compound is a PAK kinaseantisense nucleic acid or double stranded RNA molecule.
 6. The method ofclaim 1 or 2, wherein said compound is an antibody that specificallybinds a PAK kinase.
 7. The method of claim 1 or 2, wherein said compoundis an ATP analog.
 8. The method of claim 1 or 2, wherein said compoundreduces the protein level of a PAK kinase.
 9. The method of claim 1 or2, wherein said compound reduces the mRNA level of a PAK kinase.
 10. Themethod of claim 1 or 2, wherein said cancer is neurofibromatosis type 2.11. The method of claim 1, wherein said PAK kinase is human PAK2 orhuman PAK3.
 12. The method of claim 1, wherein said compound isadministered to an in vitro sample.
 13. The method of claim 1, whereinsaid compound is administered to a cell.
 14. The method of claim 13,wherein said mammal has cancer or an increased risk for cancer.
 15. Themethod of claim 2 or 13, wherein said cell is in a tissue.
 16. Themethod of claim 2 or 13, wherein said cell is in a mammal.
 17. Themethod of claim 16, wherein said mammal has cancer or an increased riskfor cancer.
 18. The method of claim 2, wherein said compound is a memberof a library of at least 5 compounds, all of which are simultaneouslyadministered to said cell.